Structure and Function of Glycosyltransferases: To date, the detailed structure-function studies on glycosyltransferases, in particular on beta1,4-galactosyltransferase-1 (b4Gal-T1) from our laboratory, have shown following: (I) Glycosyltransferases have flexible loop(s) in the vicinity of their catalytic pocket which undergo conformational changes upon donor substrate binding and create the acceptor binding site: (II) In the metal-ion dependent enzymes, the metal ion binding site is generally at the amino terminal hinge region of the flexible loop:(III) Glycosyltransferases interact with the add-on domains: To diversify the catalytic activity towards less preferred substrates, such as sugar acceptors or proteins or lipids or aglycons, the catalytic domains of glycosyltransferases either interact (1) with an additional protein, or have acquired add-on domains at the C-terminus or acquired add-on domains at the N-terminus. For example, in the lactose synthase enzyme, the b4Gal-T1, after conformational changes in the flexible loops to a closed conformation, interacts with a mammary gland-specific protein, alpha-lactalbumin (LA) at its carboxyl terminal end, changing the acceptor specificity of the enzyme towards less preferred acceptor glucose. LA protein, although not linked to b4Gal-T1, acts as an add-on domain. Several other glycosyltransferases have been shown or suggested to require an activating protein. In contrast to two interacting proteins, the catalytic domains of polypeptide a-N-Acetylgalactosaminyltransferases (ppGalNAc-Ts) have a lectin domain that is linked to at the C-terminus of the catalytic domain via a linker region and determines the specificity towards a peptide or a glycopeptide. The loops in the catalytic domain of these enzymes also undergo a conformational change upon binding of the metal ion and the sugar donor, while the lectin domain moves, bringing in the bound glycopeptide acceptor in the catalytic pocket, in order to synthesize O-a-GalNAc moiety on the glycopeptide. Also in this category is the alpha-1,6-Fucosyltransferase (FUT8), where an SH3 domain has been identified that is linked at the C-terminus of the catalytic domain. In this FY 10-11, we have now determined the crystal structure of Drosophila b4Gal-T7, which transfers Gal from UDP-Gal to an acceptor beta-xylose (bXyl) attached to side chain hydroxyl group of the Ser/Thr residue of proteoglycans synthesizing a Gal-beta-1-4Xyl disaccharide moiety. The crystal structure shows that the Drosophila b4Gal-T7 has also a flexible loop that undergoes conformational change upon binding of metal ion and donor substrate creating an accepter binding site for xylated peptide segment of a protein. (IV) A few residues in the catalytic pocket determine the donor sugar specificity of glycosyltransferases: Role of a single amino acid in the evolutionary divergence of invertebrate and vertebrate glycoconjugates: (a) Mutations in catalytic pocket of b4Gal-T1 change its donor specificity: Based on the structural information, we have previously shown, that the residue Tyr/Phe289 in the catalytic pocket of b4Gal-T1, which is conserved among all vertebrate homologs, when mutated to Leu or Ile broadens the donor substrate specificity of the enzyme to 2substituants of galactose i.e., GalNAc or 2-keto-galactose or 2-azido-galactose. In invertebrates in the b4Gal-T homologs there is an Ile residue at the corresponding position of Tyr and they are b4GalNAc-T enzymes. Mutation of the Ile residue to Tyr in Drosophila b4GalNAc-T1 converts the enzyme to a b4Gal-T1 by reducing its N-acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold.(b) Few mutations in the catalytic domain of bovine alpha-1,3-galactosyltransferase (a3Gal-T) broadens the donor specificity: We have mutated bovine a1,3-galactosyltransferse (a3Gal-T) enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives by mutating the sugar donor-binding residues at positions 280 to 282. A mutation of His280 to Leu/Thr/Ser/Ala or Gly and Ala281 and Ala282 to Gly resulted in the GalNAc transferase activity by the mutant a3Gal-T enzymes to 5-19% of their original Gal-T activity. We show that the mutants 280SGG282 and 280AGG282 with the highest GalNAc-T activity can also transfer modified sugars such as 2-keto-galactose or GalNAz from their respective UDP-sugar derivatives to LacNAc moiety present at the nonreducing end of glycans of glycoprotein, thus enabling the detection of LacNAc moiety by a chemiluminescence method. This makes it possible to use these mutants, (1) for the detection of alterations in the glycosylation patterns in many pathological states, such as cancers and rheumatoid arthritis, and (2) in the glycoconjugation and assembly of nano-particles for the targeted drug delivery of bioactive-agents. (V) The N-acetyl group of the donor sugar is generally embedded in a hydrophobic pocket of the enzyme. In both mutant enzymes,Y289L-b4Gal-T1 and SGG-a3Gal-T, the N-acetyl moiety of the donor sugar GalNAc, is embedded in a hydrophobic pocket that allows the substitution of this moiety by CH2-CO-CH3 group. We have now shown that the N-acetyl groups of the donor sugars GlcNAc and GalNAc of the N-acetylglucosaminyl- and N-acetylgalactosaminyl-transferases are generally embedded in a cavity or a hydrophobic pocket which can also accommodate a ketone group in the N-acetyl-binding pocket, making it possible to attach to the chemical handle affinity probes for detection, isolation, and characterization of the product and linking biomolecules. Galectin -1 as a fusion partner for the production of soluble and folded beta-1, 4- Galactosyltransferase-T7 in E. coli: Galectin-1 as fusion partner has been used for the expression of recombinant proteins in soluble folded and active form in E. coli. Crystal structure of the catalytic domain of Drosophila beta-1,4-galactosyltransferase-T7:Gene knockout studies in Drosophila have shown that that the b4Gal-T7, one member of the b4Gal-T family that transfers Gal to Xylose on proteoglycans, is essential for species survival while lack of b4Gal-T1 gene led to multiple disorders. However, mutations in the human b4Gal-T7 are known to cause skin fibroblasts of an Ehlers-Danlos syndrome. The catalytic domain of human b4Gal-T7 exhibits a 39% amino acid sequence similarity with the catalytic domain of human b4Gal-T1, while it shows a 68% sequence similarity with the catalytic domain of b4Gal-T7 from Drosophila. Having established crystal structure of b4Gal-T1, we have extended our crystal structure studies to include b4Gal-T7 and solved the crystal structure of the catalytic domain of b4Gal-T7 from Drosophila. Crystal structure of the catalytic domain of human beta-1,4-galactosyltransferase-T7: Having established the crystal structure of Drosophila b4Gal-T7, we are now extending our crystal structure studies on human b4Gal-T7. Since the Drosophila catalytic domain of b4Gal-T7 has a 73% protein sequence similarity to the corresponding region of human b4Gal-T7, it has offered a structure-based explanation on the effect of mutations, A186D, L206P, and R270C, the have been linked to the Ehlers-Danlos syndrome, a group of inherited connective tissue disorders. However, a disulfide bond present at the C-terminal end between Cys 255 and Cys310 is present only in the b4Gal-T7 protein from flying insects. To explain the function of this extra C-terminal sequence and the extra disulphide bond in Drosophila b4Gal-T7 we are at present determining the crystal structure of human b4Gal-T7.